Abstract LB-330: A novel role of tumor suppressor BRCA1 in regulating integrity of heterochromatin structure.

Abstract

Abstract Introduction: BRCA1, as well as BRCA2, were identified as the hereditary breast and ovarian cancer susceptibility genes that can account for almost all of inherited cases of breast cancers. BRCA1 is a multifunctional nuclear protein with a RING domain at the N-terminus. Heterochromatin is chromatin containing inactive genes. Pericentromeric heterochromatin is composed of highly repetitive tandem DNA repeats whose transcription is constitutively silenced. In mice, the repeats are classified as major and minor satellite repeats. Heterochromatin protein 1 (HP1) plays a critical role in maintaining heterochromatin structure. Methods: To study the biological function of the tumor suppressor BRCA1, we deleted BRCA1 in nestin positive neuronal stem cells in the brain and showed that the animals displayed severe neuro-developmental deficits. In the course of this work we discovered a novel molecular defect in BRCA1 deficient cells, i.e., a failure to maintain integrity of the heterochromatic structure. Results: In the absence of BRCA1, the number of heterochromatic centers is severely reduced in mouse neuronal cells. HP1 proteins are destabilized, and more importantly, the transcription of satellite DNA is derepressed in mouse neural progenitor cells, mouse fibroblasts, and human cancer cells, including a BRCA1 deficient breast cancer cell line. The defect in silencing invariably appears after the loss of the BRCA1 allele in our paradigm prior to exhibiting any neoplastic phenotype. What is the mechanism of BRCA1 in regulating the heterochromatin integrity? ChIP analysis showed that BRCA1 physically associates with both major satellite DNA and minor satellite DNA, whereas the association is diminished in the BRCA1 knockout animals. More intriguingly, the absence of BRCA1 binding to satellite DNA correlates with a drastic reduction of ubiquitinated histone H2A at the constitutive heterochromatin region. Because BRCA1 contains an E3 ubiquitin ligase activity which is enhanced by BARD1 and the complex ubiquitinate histone H2A in vitro, we examined the heterochromatin silencing in cells with different BRCA1 mutants. These experiments showed that the E3 ligase activity of BRCA1 is required for repression of satellite DNA transcription. Conclusions: Our working model is that mutations of BRCA1 may destabilize the protein complex and lead to defects in the heterochromatin integrity, thereby affecting the stability of the genome which is a hallmark of cancer. Given that mutants in the RING domains are the same as in women who have pathological diseases, we hypothesize that deregulation of satellite DNA transcription can be explored as an early detection marker for breast cancer progression in BRCA1 patients.